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Spread of P elements in drosophila melanogaster Meister, Gerald Alan 1992

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SPREAD OF P ELEMENTS IN DROSOPHILA MELANOGASTER by GERALD ALAN MEISTER B.Sc., The University of British Columbia, 1986 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA April 1992 © Gerald Alan Meister, 1992  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of  ?CO L  The University of British Columbia Vancouver, Canada  Date    DE-6 (2/88)  1 '17-  h  ABSTRACT  Several experiments suggest that P elements spread in mixed P-M populations. These experiments monitored the spread of the ability of flies to induce and to suppress gonadal dysgenesis. Such experiments, based on physiological phenotypes and providing no molecular data, are unable to distinguish between the "dispersal" and "copy number accumulation" aspects of  P element spread. This thesis describes several experiments undertaken to gain a fuller understanding of the various components of P element spread. The invasion of P elements in natural populations of Drosophila melanogaster was modelled by establishing laboratory populations with 0.5% and  5% P genomes. Two replicate populations at each frequency were monitored over twenty generations. The percentage of genomes that contained P elements was followed by single fly ovary blots at each generation for all populations. The distribution of P elements among individual flies was monitored by single fly Southern blots at even numbered generations from 6 to 20 for the two populations initiated with 5% P genomes. This analysis of the molecular dispersal of P elements is compared to the spread of the physiological phenotypes demonstrated by a collaborating lab. Our results show that the frequency of flies containing P elements increased each generation. The number of P elements within individual genomes  ii  decreased initially, but then increased to equal or surpass the number of elements in the parental P strain. Finally, the distribution of P elements within the genomes of individuals from later generations varied considerably, and this pattern differed from the original P strain. These results suggest that the interaction between the assortment and recombination of chromosomal segments, and some form of multiplicative transposition could result in the rapid spread of  P elements in natural populations.  iii  TABLE OF CONTENTS  ABSTRACT  ii  LIST OF FIGURES  vi  ACKNOWLEDGEMENTS  vii  CHAPTER I: A GENERAL INTRODUCTION TO P TRANSPOSABLE ELEMENTS IN DROSOPHILA MELANOGASTER  1  Transposable Elements; Changing Perspectives (2) Classes of Transposable Elements (3) Hybrid Dysgenesis and P elements (6) Strain Types in the P-M System (10) Structure of P Elements and the Transposase Gene (10) DNA Required in cis for Mobilization (11) Regulation of P Element Mobilization (13) Aims of this Research (16)  CHAPTER II: RAPID SPREAD OF P ELEMENTS IN EXPERIMENTAL POPULATIONS OF DROSOPHILA MELANOGASTER  iv  17  INTRODUCTION  18  MATERIALS AND METHODS  23  Drosophila Strains (23) Experimental Populations (23) Single Fly Ovary Blot Assays (24) Single Fly Southern Blots (26)  RESULTS  29  Dispersion of P Sequences Through a Population (29) Changes in P Sequences in Single Flies (37)  44  DISCUSSION Dispersal of P Activity (44) Lag in the Acquisition of P Cytotype (44) Dispersal of P Elements (49) Accumulation of P Elements and Transposition models (50) Conclusion (56)  REFERENCES  57  v  TABLE OF CONTENTS  ABSTRACT  ii  LIST OF FIGURES  vi  ACKNOWLEDGEMENTS  vii  CHAPTER I: A GENERAL INTRODUCTION TO P TRANSPOSABLE ELEMENTS IN DROSOPHILA MELANOGASTER  1  Transposable Elements; Changing Perspectives (2) Classes of Transposable Elements (3) Hybrid Dysgenesis and P elements (6) Strain Types in the P-M System (10) Structure of P Elements and the Transposase Gene (10) DNA Required in cis for Mobilization (11) Regulation of P Element Mobilization (13) Aims of this Research (16)  CHAPTER II: RAPID SPREAD OF P ELEMENTS IN EXPERIMENTAL POPULATIONS OF DROSOPHILA MELANOGASTER  iv  17  LIST OF FIGURES  FIGURE 2-1: Test of the sensitivity of the ovary blot assay  27  FIGURE 2-2: Representative ovary blots from 5% population A  30  FIGURE 2-3: Representative ovary blots from 5% population B  31  FIGURE 2-4: Percentage of females containing P sequences; 5% 33  populations FIGURE 2-5: Percentage of females containing P sequences; 0.5% populations  35  FIGURE 2-6: Distribution of P sequences in individual flies; 5% pop. A  40  FIGURE 2-7: Distribution of P sequences in individual flies; 5% pop. B  42  vi  ACKNOWLEDGEMENTS  I would like to thank Dr. T.A. Grigliatti for his patience and constructive criticism throughout the course of this work, and Dr. H.W. Brock for helpful guidance and technical advice. My gratitude also to Greg and Brenda for providing wordprocessing expertise and the use of a laser printer; Marco for assistance with figures; and Randy for assistance with graphs. Last, but not least, I thank my parents, relatives and friends for their encouragement and support.  vi i  CHAPTER I:  A GENERAL INTRODUCTION TO P TRANSPOSABLE ELEMENTS  IN DROSOPHILA MELANOGASTER  1  INTRODUCTION  Transposable Elements; Changing Perspectives: Transposable elements are discrete DNA segments that are able to move to new sites within a genome. The existence of transposable elements was first deduced by Barbara McClintock in the 1940's using purely genetic analysis. She demonstrated that there were "controlling elements" in maize that could move to new chromosomal locations and cause altered expression of nearby genes as well as chromosome breakage (McClintock 1951). Further understanding of these controlling elements would await several decades and required the elucidation of the structure of DNA, the development of recombinant DNA technology, and the discovery of transposition in bacteria. Originally, McClintock's controlling elements were thought to be rare components of the genome. Now transposable elements are considered virtually ubiquitous; they have been found in a great diversity of organisms including bacteria, yeast and other fungi, maize, fruit flies, nematodes, frogs, mice, slime molds, snap dragons, and humans (for recent reviews see Berg and Howe 1989). This remarkable mobility of both prokaryotic and eukaryotic DNA has challenged the long accepted principle that genes and their arrangement within genomes are stable and transmitted with fidelity from parents to progeny.  2  The genome of Drosophila melanogaster can be broadly separated into three categories based on reassociation kinetics: about 70% is single copy DNA, 12% is highly repetitive DNA (average reiteration frequency of about 24,000), and 12% is middle repetitive DNA (Schachat and Hogness 1974; Manning, Schmid and Davidson 1975). About 25% of this middle repetitive DNA is arranged in tandem repeats and contains genes for rRNA, 5S RNA and histones (see Spradling and Rubin 1981). The remainder of the middle repetitive DNA is comprised of 40-50 sequence families with a reiteration frequency of 35-100. The individual members of these families are found randomly interspersed with single copy sequences and they generally occupy different chromosomal locations in different strains (Manning, Schmid and Davidson 1975; Young 1979). While some of the middle repetitive sequence families of D. melanogaster are found in sibling species, many are not; closely related species of Drosophila often contain different families of repetitive elements (Dowsett and Young 1982; Dowsett 1983). These observations together suggest that these families of middle repetitive DNA, which account for about 10% of the D. melanogaster genome, are highly unstable components and are in fact transposable elements and/or remnants of transposable elements.  Classes of Transposable Elements: Sequence families of mobile elements can be further grouped according to structural similarities. These simple structural classes probably reflect the mechanisms by which the elements transpose, though much is yet to be  3  determined about these mechanisms for eukaryotic transposable elements. Most transposable elements analyzed display a variety of unique features. The classification may, therefore, represent an oversimplification. There are four broad classes of elements found in D. melanogaster (for comprehensive review, see Finnegan 1985, 1990). The largest group are the copia like elements which are named after one -  of the first elements of this type to be characterized. These are also sometimes called retrovirus-like elements due to their similarity to retrovirus proviruses. They have long terminal direct repeats and a long open reading frame that encodes a protein related to viral reverse transcriptase. At the ends of each element are short inverted repeats of about 10 base pairs and immediately before and after each element are direct repeats of several base pairs. Copia-like elements are believed to transpose by reverse transcription of an RNA intermediate via a mechanism related to the retroviral life cycle. The second class of elements are collectively termed non-viral retroposons. I elements are one of the six known members of this group which are also believed to transpose by a reverse transcription mechanism. The 3' end of one strand of each element contains an A-rich sequence and the elements are often truncated at the 5' end of this strand. They have no terminal repeats but they do encode a putative reverse transcriptase. The third class of transposable elements are characterized by long inverted terminal repeats. Perhaps the best known family of this type are called fold-back  4  elements based on their tendency to "fold-back" on themselves when allowed to reanneal at low concentration. The repeats may be found immediately adjacent to each other in the genome or may be separated by several kb of either related or unrelated DNA. The inverted repeats themselves vary in length from a few hundred to a few thousand base pairs and are not necessarily identical for any one element. These elements are thought to transpose directly from DNA to DNA. The final class of transposable elements found in D. melanogaster are those which have short inverted terminal repeats. This class includes the well characterized P elements as well as hobo, pogo and HB elements from Drosophila melanogaster. Elements from other organisms which exhibit the characteristic of short terminal inverted repeats include Ac/Ds in maize, Tam3 in Antirrhinum mcdus, Tc1 in Caenorhabditis elegans and 1723 in Xenopus laeuis (reviewed in Mobile DNA, Berg and Howe 1989). Some recent additions to this list are Uhu from Drosophila heteroneura (Brezinski et al. 1990), TECth1 from Chironomus thummi (Wobus et al. 1990), Gulliver from Chlamydomonas (Ferris 1989), and TFP3 from Trichoplusia ni (Wang, Fraser and Cary 1989). Most of these elements create an eight base pair duplication of target DNA, presumably by a staggered cut at the time of insertion. Exceptions to this generalization are Tc1, HB and Uhu which appear to insert specifically at TA sequences and create only 2 base pair target duplications (Moerman and Waterston 1989, Brezinski et al. 1990). Many of the elements share some sequence similarity over a 12 base pair region within the inverted terminal repeats (Streck, MacGaffey and Beckendorf 1986;  5  Engels 1989). These elements are all thought to transpose directly from DNA to DNA. Some comments on the various mechanisms that have been proposed for  P element transposition can be found in the Discussion (Chapter II).  Hybrid Dysgenesis and P elements in Drosophila melanogaster: The first observation of a trait that was later shown to be due to P element mobilization was made by Hiraizumi (1971). When he crossed D. melanogaster males caught in the wild to females from a laboratory stock he noted recombination in the male progeny (a trait not normally seen in Drosophila). It was later shown that the male recombination could be correlated to several other abnormal traits including temperature sensitive gonadal sterility, high mutation rate, segregation distortion and chromosome aberration (Kidwell, Kidwell and Sved 1977; Engels 1979a). Since this syndrome occurred only in the germline of the hybrid progeny of certain crosses it was called hybrid dysgenesis. D. melanogaster strains can be phenotypically categorized according to their ability to induce and/or suppress the gonadal sterility phenotype in hybrid offspring (Kidwell, Frydryk and Novy 1983). In paternal (P) strains, males can induce gonadal sterility and females can suppress it. In maternal (M) strains, males cannot induce gonadal sterility and females are susceptible. Therefore, hybrid dysgenesis occurs only when P strain males are crossed to M strain females. It does not occur in the reciprocal cross (M males to P females) or in PxP or MxM crosses (for reviews, see Bregliano and Kidwell 1983; Engels 1983).  6  The first evidence that hybrid dysgenesis was due to mobile elements came from the observation that the P factors responsible mapped to many locations in P strain genomes (Engels 1979b). The instability of certain dysgenesis induced mutations (Gulubovski, Ivanov and Green 1977; Engels 1979c; Engels 1981a) and the identification of chromosome breakage hotspots whose positions varied among chromosomes from unrelated P strains (Engels and Preston 1981) also supported the mobile element explanation for hybrid dysgenesis. The P factor hypothesis was confirmed when a transposable element insertion was found in a dysgenesis induced allele of the white locus (Rubin, Kidwell and Bingham 1982). Homologous elements were found at chromosome breakage hot spots and other dysgenesis induced mutations (Bingham, Kidwell and Rubin 1982). All of the traits associated with hybrid dysgenesis have since been shown to be in some way associated with an elevated level of P element activity in hybrid progeny. The increased mutation rate and chromosome rearrangements are a direct result of an increased level of the transposition, excision, and insertion of P elements within the genome. Many P element insertional mutants have been identified and it has been estimated that at least half of the spontaneous mutations in D. melanogaster are due to the insertion of various mobile elements (Engels 1989; Finnegan 1990). Engels and Preston (1984) suggest that the chromosome rearrangements could result from the simultaneous mobilization of two or more P elements. The excision of the elements could cause chromosome breakage at several locations followed by subsequent reunion of incorrect pieces.  7  Dysgenic sterility and transmission ratio distortion are less directly related to the movement of P elements. Dysgenic sterility is characterized by a failure of the gonads to develop properly and is therefore often called gonadal dysgenesis or GD sterility (Kidwell and Novy 1979; Engels and Preston 1979). It is thought to be the result of dominant lethal mutations that kill cells in the developing germ line and prevent the formation of functional gonads (Simmons et al. 1987). Transmission ratio distortion refers to the tendency for progeny of dysgenic flies to carry chromosomes derived from the P strain male rather than the M strain female. The amount of distortion against an autosome increases with the number of P elements it carries (Rasmusson et al. 1990). It is likely that transmission ratio distortion occurs when chromosomes that are broken by P element mobilization are lost because of the inviability of the gametes that contain them (Simmons et al. 1987). Establishing the relationship between P element activity and hybrid dysgenesis-induced recombination has proven to be difficult. There are a number of observations that need to be explained. Although originally discovered because of recombination induced in males, P element induced recombination has been shown to occur in females as well (Kidwell 1977). The chromosomal distribution of the induced recombination clearly differs from the normal meiotic recombination seen in females; the distribution of induced recombination more closely fits the polytene chromosome map (Kidwell and Kidwell 1976; Sinclair and Grigliatti 1985). However, dysgenesis-induced exchanges do not occur in  8  heterochromatic regions, and there are marked strain-specific expansions and contractions of certain regions (Sinclair and Grigliatti 1985). It has been demonstrated that induced male crossing over occurs in genetic regions containing an incomplete element (Sved, Eggleston and Engels 1990). However, it has also been shown that a single non-mobile P element, which provides a source of the element encoded transposase protein, is able to induce male recombination (McCarron et al. 1989). This male recombination occurs even in the absence of other P elements which might be mobilized in response to the transposase protein. A more recent study from the same lab found no correspondence between mobilization of P elements and recombination within specific intervals (Duttaroy  et al. 1990). These results suggest that transposase may be active at non-P element sites even when potentially mobile P elements are present. This latter report also showed that two short adjacent regions had very different male recombination frequencies despite a lack of insertion or excision activity. They interpret this to mean that there may be regions of the genome which are inherently more or less responsive to transposase induced recombination. They further hypothesize that P elements may act as a focus for induced recombination by attracting transposase to the chromosomal region. Once bound to the chromosome the transposase action may radiate out to work on genomic sequences over some distance.  9  Strain Types in the P-M System: There is considerable variation in the P-M phenotype besides the P and M strain extremes. Strong P strains have 30-50 copies of P sequences, but as few as 30% of these may be complete P factors (O'Hare and Rubin 1983). The complete  P factor is 2.9 kb long while shorter P elements contain internal deletions and range in size from 0.5 to 2.9 kb (Rubin, Kidwell and Bingham 1982). There are two main subtypes of M strains. True M strains completely lack P elements by molecular analysis and have extremely high susceptibility to P strains (Kidwell 1985); pseudo M (M') strains contain P elements, sometimes many of them, and their susceptibility to hybrid dysgenesis ranges from low to high (Engels 1984; Boussy and Kidwell 1987). Neutral or Q strains are defined as strains that cause less than 10% gonadal sterility among hybrids with M strain females but are also resistant to the dysgenic activity of P strain males (Kidwell and Novy 1979; Kidwell, Frydryk, and Novy 1983). Q strains may have only a subset of P sequences, or may have a reduced number of transposase producing P elements relative to P strains (Bingham, Kidwell and Rubin 1982).  Structure of P Elements and the Transposase Gene: The complete P element, sometimes called the P factor, is 2,907 base pairs long (O'Hare and Rubin 1983). Its sequence reveals several interesting repeat structures. The termini contain 31 base pair (bp) perfect inverted repeats and  10  about 125 by from each end there are 11 by inverted repeats. Internally, within the coding region, there are slightly separated 17 by inverted repeats and a 20 by overlapping direct repeat. The possible importance of the first two repeat structures is discussed below; there is no known functional significance for the last two. In germline tissues, the complete element expresses transposase, an 87 kd protein required for transposition and excision (Bingham, Kidwell and Rubin 1982; O'Hare and Rubin 1983; Rio, Laski and Rubin 1986). The P element has four open reading frames (ORF 0, 1, 2, and 3) and mutational analysis has revealed that all four are required in cis to encode a functional transposase protein (Karess and Rubin 1984; Laski, Rio and Rubin 1986; Rio, Laski and Rubin 1986). Incomplete P elements are very heterogeneous but can be derived from the full sequence by internal deletions (O'Hare and Rubin 1983). The incomplete elements do not encode a functional transposase but can be mobilized in the presence of complete P factors (Spradling and Rubin 1982; Rubin and Spradling 1982). The complete and incomplete elements are therefore sometimes referred to as autonomous and nonautonomous elements respectively.  DNA Required in cis for Mobilization: It has long been known that proper termini are required for P element mobilization (Rubin and Spradling 1983; Karess and Rubin 1984). Mullins, Rio  11  and Rubin (1989) used germline transformation of a variety of modified elements to determine the precise cis requirements for mobility. They found that about 150 by of DNA from each end of the P element was essential. This region includes the 31 by terminal inverted repeats, unique sequences at the 3' and 5' ends, and the internal 11 by inverted repeats. The P element transposase protein has recently been purified and characterized (Kaufman, Doll and Rio 1989). It is a site-specific DNA binding protein that specifically interacts with a 10 by consensus sequence that is located just internal to the terminal repeats at both ends of the P elements. These sequences lie within the regions shown to be important for transposition. On the 5' end the binding site overlaps sequences shown to be essential for P element transcription. This raises the possibility that transposase (or the 66 kd P element protein described below) could regulate transcription from the P element promoter. Transposase protein does not interact with the inverted repeats. This may suggest that the transposition reaction requires the binding of additional Drosophila protein factors to the P element termini. Requirements for hostencoded functions are common for prokaryotic transposition reactions (for reviews see Berg and Howe 1989). One host encoded protein that interacts specifically with the outer 16 by of both terminal repeats of the P element has been found (Rio and Rubin 1988).  12  Regulation of P Element Mobilization: There are at least three types of gene regulation associated with P element mobilization. First, P element transposition is restricted to germline cells. Second, the reciprocal cross differences that are characteristic of hybrid dysgenesis are regulated by a cellular condition, known as P cytotype. Finally, P element activity is suppressed by M' strains. While P elements can transpose at high frequencies in the germline of certain hybrid flies, they are almost completely stable in somatic tissue. This tissue specificity is regulated at the level of pre-mRNA splicing of the intron between ORF 2 and ORF 3 of the P element (Laski, Rio and Rubin 1986). The mature RNA in somatic tissue retains the third intron and gives rise to a smaller 66 kd protein instead of 87 kd transposase. Laski, Rio and Rubin put a modified element, lacking the third intron, into the genome. They found high levels of somatic activity as indicated by somatic mosaicism for P element induced mutations. There is now evidence that the 66 kd protein can act as a negative regulator of transposition (Robertson and Engels 1989; Misra and Rio 1990). While the above results establish the 66 kd protein as a repressor of transposition in somatic cells, it raises the question of how the alternative splicing is controlled. Siebel and Rio (1990) have demonstrated that the third intron can be spliced by a heterologous cell extract but that this splicing was blocked by  13  preincubation of the pre-mRNA with Drosophila somatic cell extract. Using UV cross-linking they went on to show that a 97 kd Drosophila protein present in the cell extract binds preferentially to sequences near the 5' splice site. This binding can be correlated with the inhibition of splicing in vitro. This evidence supports a model in which specific proteins in somatic cells block the splicing of the third intron resulting in the absence of functional transposase and the production of a 66 kd repressor of transposition. The nonreciprocal nature of the hybrid dysgenesis phenomena is due to a regulatory ability of P strain females which is manifested in their eggs. Individuals that are resistant to the action of P elements have been described as having P cytotype, whereas those that are susceptible have been said to have M cytotype (Engels 1979b). Hence, hybrid dysgenesis occurs only when P elements are present in an embryo with M cytotype. Cytotype is bimodal; flies with mixed P and M ancestry usually display either an M or P cytotype rather than some intermediate state (Engels 1979b). The cytotype of a fly is determined by an unusual mode of inheritance that involves both the individual's genotype and the mother's cytotype. The frequency of P cytotype is higher in the progeny of mothers with P rather than M cytotype and this frequency increases with the number of P factors in the genome (Engels 1989). In the absence of P elements the cytotype is always M. The molecular models proposed to account for cytotype regulation of hybrid dysgenesis are discussed in detail elsewhere (Chapter II Discussion).  14  The partial suppression of hybrid dysgenesis exhibited by most Q and M' strains differs in two ways from that exhibited by P strains. First, it is only chromosomally inherited and therefore is fully transmissible through the male line (Kidwell 1985; Simmons and Bucholz 1985; Black et al. 1987). Second, instead of the bimodality associated with cytotype, various combinations of M and M' chromosomes result in all levels of dysgenesis when crossed to P strain males (Kidwell 1985; Simmons and Bucholz 1985). The M' type suppression may result in part from the presence of certain deleted elements, KP elements, which are present in high copy number in some M' strains (Black et al. 1987). Some genetic evidence for a regulatory role for KP elements was presented, however, in this study the possible effects of other P elements were not separated from the effects of KP elements. Also, at least one M' strain suppresses gonadal dysgenesis, but contains no KP elements (Black et al. 1987; Boussy et al. 1988). This is further complicated by data from Black et al. (1987) showing that some strains contain KP elements but do not suppress GD sterility. Some of the suppression exhibited by M' strains can be explained by titration of transposase (Simmons et al. 1984; Simmons and Bucholz 1985). The titration model suggests that, if P element termini act as a substrate for transposase during transposition, then the presence of many nonautonomous elements might leave less transposase to act on particular elements. This would lead to a decrease in mutations associated with P element mobilization. However, there would be an increase in GD sterility, since mobilization of nonautonomous  15  elements probably contribute to this trait. This is exactly the result Simmons and Bucholz obtained when they increased the number of M' chromosomes in the genome. Thus, the titration model may explain some aspects of the M' type regulation of P element mobilization. However, it fails to explain the partial suppression of GD sterility exhibited by certain M' strains.  Aims of this Research: Clearly the hybrid dysgenesis phenotype is complex. In recent years much has been learned about the structure of the P element, its tissue-specific regulation at the level mRNA splicing, and the mechanisms by which P elements induce the various components of the hybrid dysgenesis phenomena. Much less is known about the molecular biology of cytotype repression, the mechanisms of excision and transposition, and the rate and possible specificity of P element deletion. Also, while the spread of the phenotypes associated with P elements has been studied in mixed populations, little is known about this spread on a molecular level. This thesis examines the dispersal and accumulation P elements on a molecular level within individual flies of mixed P-M populations. These data are correlated with the spread of the dysgenesis phenotype.  16  CHAPTER II:  RAPID SPREAD OF P ELEMENTS IN EXPERIMENTAL POPULATIONS OF DROSOPHILA MELANOGASTER  17  INTRODUCTION  Based on theoretical considerations, it has been argued that the "spread" of multi-copy elements, such as P elements, in mixed populations involves several components; namely, the invasion of genomes that lack elements, an increase in element copy number within genomes, and competition between complete and deleted elements (Hickey 1982; Ginsburg, Bingham and Yoo 1984; Kaplan, Darden and Langley 1985). In the specific case of P elements the spread of the regulatory ability, called P cytotype, must also be considered. The spread of elements in mixed populations is of considerable interest for several reasons. First, there is the academic issue of whether elements can actually spread quickly through a population. This may help in understanding the distribution of elements amongst various natural populations and laboratory populations (reviewed below). A second issue is whether transposable elements might have a practical application. One such application may be as transformation vectors. An extension of this application might be to utilize these vectors to disperse engineered constructs through naive genomes. This could be utilized to spread a gene that is detrimental to a pest insect or a gene that somehow increased the productiveness of a beneficial insect.  18  Considerable variation in the P-M phenotype exists amongst worldwide populations of Drosophila melanogaster. Surveys of wild populations from America, Japan, Europe, Asia, Africa and Australia reveal these populations to be P, Q or M' (Anxolabehere, Nouaud and Periquet 1982; Bregliano and Kidwell 1983; Kidwell, Frydryk and Novy 1983; Kidwell 1983; Takada et al. 1983; Yamamoto, Hihara and Watanabe 1984; Anxolabehere et al. 1984, 1985; Kidwell and Novy 1985; Boussy 1987; Boussy and Kidwell 1987; Anxolabehere, Kidwell and Periquet 1988). Strains from a given geographical region are often predominantly of one type, however, Q strains can be found in virtually all geographic regions (Engels 1989). Several broad patterns have been established. The frequency of Q strains gradually declines from west to east in Europe and central Asia (Anxolabehere et al. 1985). Eastern Australia populations can be divided into three regions based on the predominant strain types which change over short distances from P to Q to M' in a north to south direction (Boussy 1987; Boussy and Kidwell 1987; Boussy et al. 1988). Such P-M variation suggests that P elements are not at equilibrium in wild populations of Drosophila.  Nevertheless, all strains derived from natural populations since 1974 have been shown to contain at least some P elements (Anxolabehere, Kidwell and Periquet 1988; Boussy et al. 1988). In contrast to natural populations, long-established laboratory strains are usually true M type (Bingham, Kidwell and Rubin 1982; Kidwell 1983; Kidwell, Frydryk and Novy 1983; Bregliano and Kidwell 1983). Two hypotheses have been  19  proposed to account for this difference between lab and natural populations. The stochastic loss hypothesis (Engels 1981b) suggests that P elements have always been present in substantial frequencies in natural populations, and that their absence from long-established laboratory populations is due to loss of P elements from these strains by genetic drift. In contrast, the recent invasion hypothesis (Kidwell 1983) posits that P elements did not exist among natural populations prior to the 1950's and that P sequences recently invaded natural populations of Drosophila melanogaster, spreading rapidly by replicative transposition. A  prerequisite to the recent invasion hypothesis is that P elements must be able to rapidly invade true M populations. Several experiments have monitored the phenotypes of gonadal sterility and P cytotype in mixed P-M populations. Kidwell, Novy and Feeley (1981) showed that, in the absence of measurable gonadal dysgenesis (20°C), mixed populations changed unidirectionally toward P type. Kiyasu and Kidwell (1984) found that most mixed populations also evolved to P type under conditions of strong sterility (27°C). These observed increases in the frequency of P type flies provide strong evidence that P elements can spread once introduced into a true M population. The use of the dysgenesis phenotype alone as a marker to monitor the spread of P elements is complicated by the fact that there are multiple dispersed copies of these elements within each genome. P strains usually contain 30 to 50 elements per individual. It is possible that only a few elements may be required to induce and/or suppress hybrid sterility. Mixed population experiments which  20  monitor only physiological phenotypes can therefore only make inferences about the dispersal of elements to new genomes and the accumulation of elements within populations or within individuals. Some recent experiments have utilized Southern blot or in situ hybridization analyses to demonstrate that P elements can accumulate within individual genomes of inbred lines over several generations (Daniels et al. 1987; Preston and Engels 1989). These inbred lines were established from flies which had acquired one or more elements by transformation. Results obtained from such inbred lines are not applicable to natural populations. Furthermore, these experiments cannot demonstrate the dispersal of elements to new genomes since all flies used to initiate the populations contain at least one element. This chapter describes several experiments undertaken to gain a fuller understanding of both the "dispersal" and "copy number accumulation" aspects of P element spread. The invasion of P elements into natural populations that completely lack P elements was modeled by the introduction of low frequencies (0.05 and 0.005) of P genomes into true M populations. The experiments were carried out at 23°C so that GD sterility would not occur. The dispersal of P elements to new genomes was followed at each generation by single fly ovary blots. In addition, single fly Southern blots were used to monitor both the accumulation and the distribution of elements within individual genomes. A collaborating lab set up similar experimental populations and monitored the spread of P phenotypes in subsequent generations (Good et al. 1989). Specifically,  21  they tested flies for the acquisition of the ability to induce and to suppress hybrid sterility. To confirm the parallelism between the two sets of experiments they monitored the accumulation of P element DNA within their populations. This was done by performing dot blots on DNA extracted from multiple flies at generations 5, 10, 15 and 20. The results of the molecular assays presented in this chapter were correlated with the physiological data from the collaborating lab after both sets of experiments were completed.  22  MATERIALS AND METHODS  Drosophila Strains: (1) 72: An inbred wild type P strain originally collected from Madison, Wisconsin by W.R. Engels (Engels 1979b). (2) Canton-S: A wild type laboratory true M strain that was used as the M stock.  Experimental Populations: Experimental populations were maintained at 23 °C on yeast-sucrosecornmeal agar medium to which tegosept (methyl-p-hydroxybenzoate) was added as a mould inhibitor. Populations were initiated with mated Canton-S (true M type) females only. A proportion (1% or 10%) of these females were mated with P type males, the remainder were mated with Canton-S males. In this way we could introduce a small proportion of P genomes while minimizing the effects of genetic drift and fertility differences in the first generation of the experiment. Each population was established by placing 1000 mated females into 25 half pint bottles; two replicate populations of the two initial P frequencies were started. These populations are henceforth referred to as 5% and 0.5% populations A and B based on the percentage of P genomes in the founding flies. Every generation the flies from each of the four populations were collected, pooled, and  23  mixed. Then about 60 flies were distributed to each of 25 new bottles to establish the next, discreet generation. After several days the parents were discarded. Presence of P elements was monitored at each generation by means of single fly ovary blots. In addition, at even numbered generations between generations 6 and 20, samples of flies were quick frozen by placing them in eppendorf tubes and subjecting the tubes to an ethanol/dry ice bath. These samples were stored at -70°C. Genomic DNA from single flies could then be analyzed by Southern blot hybridization to determine the distribution of P elements within individual genomes.  Single Fly Ovary Blot Assays: Ovary blot assays were done by a technique similar to the one used to study the distribution of P sequences in natural populations (Anxolabehere et al. 1985). Nitrocellulose filters were equilibrated on 2 thicknesses of Whatman 3MM filter paper that had been soaked in 5% SDS, 0.05 M EDTA. Ovaries were dissected from females onto the nitrocellulose. After 30 min, the nitrocellulose filters were floated on a 0.5 ml "puddle" of 0.5 M NaOH for 7 min to denature the DNA. The filters were then neutralized by soaking them twice in 1 M Tris-HC1 (pH 7.4) for 2 min each. Finally, the filters were treated for 4 min with 1.5 M NaC1, 0.5 M Tris-HC1 (pH 7.4), air dried and baked for 2 h at 80°C. The filters were wrapped in foil and stored so that several generations could be probed at once. This minimized variation due to the amount and intensity of the probe.  24  Pre-hybridization and hybridization were carried out under conditions modified from Maniatis, Fritsch and Sambrook, 1982. About 3 ml per filter of prewarmed prehybridization solution (50% formamide, 5X SSC, 50 mM sodium phosphate, 2X Denhardt's, 100 µg/ml sheared denatured salmon sperm DNA, 0.1% SDS) was added to filters in bags which were then heat-sealed and incubated for 3 h at 42°C. The 0.84 kb HindIII fragment ofp725.1 (Spradling and Rubin 1982; Karess and Rubin 1984) was nick-translated, filtered through a Sephadex G-50 spin column to remove unincorporated nucleotides (Maniatis, Fritsch and Sambrook 1982), and denatured. About 'Ong of probe DNA per ml of prehybridization solution was added to the bags. This P element fragment encompasses nucleotide positions 39 to 877 of the complete element. It was chosen because it should hybridize to the majority of P elements, both complete and internally deleted (O'Hare and Rubin 1983). After hybridization at 42 °C for about 14 h, the filters were washed 4 times for 10 min each in 3 mM Tris -HC1, 0.1% SDS and autoradiographed. Ovaries from 72 (P) and Canton-S (true M) flies were included on each filter as controls. The amount of probe bound to ovary DNA from experimental flies was scored visually as positive or negative with respect to these controls. Any questionable dots were scored as negative. No attempt was made to quantify the intensity of the signal. To test the sensitivity of this assay, ovaries from several strains with known numbers of P elements were examined. Among the strains tested were tAP-1, tAP-3, tAP-5, and tAP-14 (Goldberg, Posakony and Maniatis  25  1983). Each of these strains is homozygous for a single, stable P element with an  Adh insert. The ovaries from females of all three of these strains, as well as any strains with more elements, always tested positive when compared to the Canton-S controls (see examples in Figure 2-1). Thus, it appears that this method can routinely detect an individual with only two P elements. Presumably this method can detect equally well those flies heterozygous for two elements at distinct loci and those flies homozygous for an element at one locus.  Single Flv Southern Blots: Each fly was homogenized in 100 pl of 0.125 M Tris-HC1 (pH 8.5), 0.08 M NaC1, 0.05 M EDTA, 0.16 M sucrose, 0.5% SDS, and incubated for 30 min at 65°C. Potassium acetate was added to a final concentration of 1 M, and the solution was kept on ice for 60 minutes. After centrifugation for 10 min in a micro-centrifuge at 4°C, the supernatant was extracted with an equal volume of equilibrated phenol/chloroform. The nucleic acids were precipitated by addition of 2.5 volumes of ice cold 95% ethanol and 15 min centrifugation. The pellet was then rinsed with cold 70% ethanol, dried briefly in a vacuum desiccator, and resuspended in 10 pl of solution containing BamH1, XbaI and the appropriate buffer. This solution was made to 50 µg/ml with RNase and digestion was allowed to proceed for 2 h at 37°C. The samples were then loaded onto 0.7% agarose gels and separated electrophoretically. Blotting to nitrocellulose membrane was carried out overnight in 10X SSC. The filters were air dried and then baked for  26  1 2 • 3 • 4 5  • • . • • • • • • • . • * •  as  M P  TEST I C  FIGURE 2-1: Test of the sensitivity of the ovary blot assay. The right hand column labelled C (for controls) contains ovaries from two Canton-S (true M strain) and two 72 (P strain) females. The rest of the filter is divided into five rows, each containing five ovaries from a different test strain. For rows 1 to 5 respectively these strains are tAP1, tAP-3, tAP-5, tAP-14, and Harwich. Each of these test strains is homozygous for a single P element except for Harwich which is a strong P strain.  27  2 h at 80°C. Prehybridization and hybridization were done via the methods of Maniatis, Fritsch and Sambrook, 1982. Filters were prehybridized for 3 h at 65 °C in 6X SSC, 0.5% SDS, 5X Denhardt's solution and 100 µg/ml denatured salmon sperm DNA. The nick-translated 0.84 kb Hind III fragment of pr25.1 was added and hybridization allowed to proceed for approximately 14 h at 65 °C. Filters were washed for two times 30 min in 2X SSC, 0.1% SDS and then for 10 min in 0.1X SSC, 0.1% SDS, and an autoradiogram was made. The P element probe was then completely removed by washing the filters twice for 1 h in 500m1 of 5mM Tris-HC1 (pH 8.0), 0.2mM EDTA (pH 8.0), 0.02% sodium pyrophosphate, and 0.1X Denhardt's solution. Removal of the probe was checked by autoradiograph. Filters were then prehybridized, re-hybridized to sAC-i (an Adh single copy probe; Goldberg 1980), and washed under the same conditions utilized for the P element probe. Another autoradiogram was made so that the amount of DNA loaded in the various lanes could be compared.  28  RESULTS  Dispersion of P Sequences Through a Population: The rapid "spread" of the P strain characteristics of induction and suppression of gonadal sterility has been demonstrated in mixed populations established using the same strains as the ones in this thesis (Good et al. 1989). This spread of P phenotype is expected to be correlated with an increase in the frequency of genomes containing P sequences in my populations. The proportion of P element containing flies in successive generations of each population was estimated using single fly ovary blots (see Materials and Methods). At each generation the ovaries were removed from 200 individual female flies and the DNA from each was isolated on nitrocellulose filters. These filters were probed with an internal P element fragment. The hybridization signal of the experimental flies was compared to the signal obtained with reference true M (Canton-S) and P (2-2) strain flies, and scored visually as positive or negative. Figures 2-2 and 2-3 show typical results from generations 1,3,6,9,15 and 20 of 5% populations A and B respectively. Clearly there is an increase in genomes containing P elements with increasing generation for both populations. While this method is quite sensitive, it is likely to underestimate the number of flies containing P elements, since dubious signals were interpreted as negative.  29  •  ENE  • •  •  •  • •  15  • •  	.•	 • • • • •  •••••  •  • • . • • • • • 04  • ••••  •••••_ • • • • • • • • • •••  20  ••••• •• •••• •••• M  • ••41101p •••••*.  FIGURE 2-2: Representative ovary blots from generations 1, 3, 6, 9, 15 and 20 of 5% population A. Each blot has the ovary DNA from 25 experimental females (labelled E on last blot) and all were hybridized simultaneously to an internal P element probe. The right hand column of each blot (labelled C) has the ovaries of two true M strain (Canton-S) and two P strain (72) control females.  30  1  3  6  •  •  t • •  •  ••  •  • • ••••  9  • • •  ••••  I • • • • _ t • •• dot 04 • • • •• •  •  15  20  •••.•• _ . . f•••tf  OD • • • • • lib  _  •• • • • eil•  . i  •• •  d•••  a ••••  M  • •• • •  ••••• • p • a • •  -  FIGURE 2-3: Representative ovary blots from generations 1, 3, 6, 9, 15 and 20 of 5% population B. Each blot has the ovary DNA from 25 experimental females (labelled E on last blot) and all were hybridized simultaneously to an internal P element probe. The right hand column of each blot (labelled C) has the ovaries of two true M strain (Canton-S) and two P strain (-/r2) control females.  31  The percentage of flies containing P elements at each generation from 5% populations A and B are shown in Figure 2-4, panels A and B respectively. The number of flies that have P elements increased rapidly in both populations until about generation ten, at which point approximately 90% or more of the individuals contained a detectable number of P sequences. Then the rate of P element spread diminished. Figure 2-5, panels A and B, show the percentage of flies containing P elements at each generation for the 0.5% populations. The frequency of P element containing flies increased slowly in both populations until about generation 6 when about 15% of the flies contained elements. At this point population A underwent a rapid increase in P element containing genomes; over 60% of the females from this population tested positive by generation 10. The increase in P element containing flies in 0.5% population B lagged for several generations. A rapid increase appeared to start in generation 10, the last generation for which results are available. Unfortunately both of these populations produced very few adult progeny from generation 11 onward. Although eggs were laid and the larvae appeared to develop normally, the pupae turned black and the flies did not eclose. This occurs periodically in Drosophila cultures and may be due to such things as the pupae becoming too dry or the population becoming infected with some parasite. We do not believe that the deaths were related to the activity of P elements. Despite the premature end to these 0.5% P populations, it is clear that the P elements were not lost and that they were able to spread to a significant number of genomes. Several more mixed  32  FIGURE 2-4: Percentage of females containing P sequences; 5% populations.  P sequences were detected by probing ovary blots with an internal P element fragment. Panels A and B summarize the results of replicate populations initiated with 5% w2 genomes.  33  A  5% Population A 120 100 80 60 40 )#(  20  o  )n(  o  2 4 6 8 10 12 14 16 18 20 22 GENERATIONS  5% Population B 120 100 80  *  *  )1(  60 Ww  40 )4(  20  o o 2 4 6 8 10 12 14 16 18 20 22 GENERATIONS  34  FIGURE 2-5: Percentage of females containing P sequences; 0.5% populations.  P sequences were detected by probing ovary blots with an internal P element fragment. Panels A and B summarize the results of replicate populations initiated with 0.5% 72 genomes.  35  A    0.6436 Population A  70 mg  00  s_ es  60  a  140  at  30  20  me  ^  1 0  Mt  Mt  0 0  i  Wt    1  i  2  3  4  6  6  7  8  9 10 11  KKKKK AT 10 Ni  0.596 Population B  70  00  60 es 6 !E *40 ss ez, co M 30 :s se 20 Mt  MI SE  IN  1 0     St  2  3  04  0 0  )1(  y 1  4  6  0  7  OE Ild SR AT ION IP  36  8 9 10 11  populations utilizing different P and M strains have been established. Ovary blots demonstrate that, in some of these populations, P elements were capable of spreading from a starting frequency of 0.5% to virtually all of the progeny within 12 to 15 generations (unpublished data). These results show that, if a few P flies are introduced into a randomly mating true M population, the spread of P elements to new genomes can be very rapid. While no attempt was made to quantify the ovary blots, the intensity of the signals is probably a rough reflection of the number of elements within each fly. Note that a single probe was hybridized simultaneously to all filters shown in Figures 2-2 and 2-3, and also that the w2 controls all have a similar intensity. In the early generations the average intensity of hybridization to the ovaries decreases with increasing generation (compare generations 1, 3 and 6). However, in later generations this trend is reversed. From generation 9 onward the positive signals are not only more frequent, but also, they appear more intense. This observation was made while testing large numbers of ovaries (200 flies for each generation of four populations) and suggests an increase in the number of P sequences per fly in later generations.  Changes in P Sequences in Single Flies: The results obtained above (and by Good et al. 1989) indicate both a dispersion and multiplication of P element sequences. To confirm these results, single fly Southern blots were performed. DNA was prepared from single flies of  37  even numbered generations from generation 6 to 20 of the two populations initiated with 5% w2 genomes. The DNA was digested with BamH1 and XbaI, separated by electrophoresis, transferred to nitrocellulose filters, and probed with an internal P element fragment. Since neither of the two restriction enzymes used to cut the genomic DNA cut within the complete P element, one would expect this combination of enzymes and probe to yield one band for each hemizygous or homozygous P element. Figures 2-6 and 2-7 show Southern blots containing DNA of flies from generations 6, 14 and 20 of 5% populations A and B respectively. The blots also contains the DNA of the Canton-S (true M) and 72 (P) reference strains. Notice that the 2-2 controls did not yield as many bands as might have been expected. In situ hybridization reveals the w2 strain to have about 30 P elements per genome (Bingham, Kidwell and Rubin 1982) yet only about 12 bands are visible in the 72 control lane of the gel. This result may be partially due to the use of a small internal P element fragment as a probe. However, this fragment is very close to one end of the P element. Since incomplete elements generally arise by internal deletion of the complete P element, this fragment should hybridize to most of them. Similar incomplete detection of P elements has been found by others even with multiple fly Southern blots. Daniels et al. (1987) detected about 40 bands in Southern blots of Harwich-77 when in situ analyses had detected 60 to 65 elements. Blackman and colleagues (1987) also show far fewer P element hybridizing bands in Southern blots of 72 than expected. This simply implies that the actual number of elements  38  per fly will be underestimated using Southern blot analysis. Co-migration of bands may be partially responsible for this result since a large number of elements are present in P genomes. Despite the fact that the single fly Southern blots may underestimate the actual number of elements per fly, several observations can be made from Figures 2-6 and 2-7. First, the number of P element homologous sequences per individual increases dramatically between generations 6 and 20. Second, the intensity of labelling and the number of bands in generation 20 resemble or are greater than the P strain control. Finally, in later generations, many of the bands are not shared either between individuals of a given generation or with the original P strain. The first two observations demonstrate that P sequences are increasing in number within individual flies. The three observations together suggest that the P elements are multiplying and transposing.  39  FIGURE 2-6: Distribution of P sequences in individual flies; 5% population A. DNA from single flies was digested with BamH1 and XbaI, Southern blotted and probed with an internal P element fragment. Panel A shows results for generations 6, 14 and 20 of 5% population A. A Canton-S and a 12 lane are included as true M and P controls, respectively. The blot was washed and reprobed with an Adh probe. Panel B shows the hybridization obtained to a unique BamH1-XbaI fragment of approximately 5 kb in length, located 5' of the Adh gene (starting at nucleotide -660). This can be used to compare the amount of DNA loaded into individual lanes.  40  6   1    14 1  20  MP 1  A  fo••••  4110414a • 40.4.4, 4,  B  41  1111. 110  FIGURE 2-7: Distribution of P sequences in individual flies; 5% population B. DNA from single flies was digested with BamH1 and Xbal, Southern blotted and probed with an internal P element fragment. Panel A shows results for generations 6, 14 and 20 of 5% population B. A Canton-S and a 72 lane are included as true M and P controls, respectively. The blot was washed and reprobed with an Adh probe. Panel B shows the hybridization obtained to a unique BamH1-XbaI fragment of approximately 5 kb in length, located 5' of the Adh gene (starting at nucleotide -660). This can be used to compare the amount of DNA loaded into individual lanes.  42  PM  6  14   I  20  111 LI I III  11 a .  40.  V PM  A  • go ao al• 4  4111111111411 SD 41. MO  B  43  4111 4114  Oa  DISCUSSION  Dispersal of P Activity: These experiments model the introduction of low frequencies of P flies into randomly mating true M populations. In similar populations it has been demonstrated that, with increasing generation, there is a very rapid increase in the amount of dysgenesis induced by a sample of flies (Good et al. 1989). This data confirmed earlier results and suggested that P elements can rapidly spread in mixed P-M populations in the absence of any imposed selection (Kidwell, Novy and Feeley 1981; Kiyasu and Kidwell 1984; Kidwell 1986).  Lag in the Acquisition of P Cytotype: The acquisition of P cytotype in the populations, as measured by the ability to suppress gonadal dysgenesis, occurred markedly later than the increase in the ability of flies to induce gonadal dysgenesis (Good et al. 1989). These results support earlier observations that the M cytotype will often be retained for some time in the presence of P sequences, but that eventually the cytotype will change (Kidwell and Novy 1979; Engels 1981a; Kidwell 1986). Any model proposed for P cytotype must be compatible with this observed lag in cytotype switching.  44  One frequent suggestion is that P cytotype is due to a repressor of P element activity; this repressor would be encoded by the elements themselves and production would in some way be dependent on the cytotype of the individuals mother (Engels 1981a; O'Hare and Rubin 1983). Since all 4 major exons are required to encode the transposase protein, such a repressor must either be very small or else must overlap with the transposase. The latter possibility is particularly inviting since this is the case with the regulation of certain other elements such as the prokaryotic transposon Tn5 (Berg 1989) and the maize transposable element Spm (Masson et al. 1989). Overlapping genes for transposase and a repressor would suggest some possible mechanisms of repression based on the two proteins sharing certain domains. For example the repressor might compete with the transposase for binding sites on the P element termini, or might form a nonfunctional transposase complex with host proteins. In either of these scenarios an obvious candidate for the repressor gene would be certain incomplete P elements. The generation of these elements may be specific; however, this is not a requirement. Several incomplete elements that are capable of repression of hybrid dysgenesis have been described. Nitasaka, Mukai and Yamazaki (1987) have demonstrated that a chromosome segment that repressed transposition contained two incomplete P elements. They dismissed one as too small to be an important regulatory element, and showed that the other was a deleted P element carrying only open reading frames 0 through 2. Robertson and Engels (1989) have shown that the presence  45  of a single element mutated in or near the 2-3 intron or within open reading frame 3 can also substantially reduce hybrid dysgenesis, though this regulation lacks the reciprocal cross effect associated with cytotype. They hypothesize that chance deletions produce elements that code for a repressor rather than a transposase. These findings are consistent with the hypothesis that the 66 kd protein produced by a lack of splicing of the third and forth transposase exons can act as a repressor in somatic tissues (Rio, Laski and Rubin 1986; Robertson and Engels 1989; Misra and Rio 1990). Generation of a similar protein in germ cells by a modified element may be responsible for P cytotype. Indeed, Misra and Rio (1990) have shown that an authentic P strain produces high concentrations of a 66 kd protein during oogenesis. If the induction of hybrid dysgenesis is dependent on the presence of P factors, and P cytotype is dependent on certain incomplete P elements, then two models could plausibly explain why acquisition of P cytotype lags behind the ability to induce gonadal dysgenesis. The simplest model is that the number of  P factors within the genomes of the invading flies is substantially greater than the number of cytotype generating P elements. Outbreeding with true M flies would produce some progeny containing P factors, but lacking cytotype generating  P elements. Alternatively, the lag in cytotype switching could be explained if P factors undergo replicative transposition at higher rates than the cytotype generating P elements. These models are not mutually exclusive. In either case, new cytotype determining elements might have to be generated, and this process  46  might take many generations, especially if the process is not specific. Degenerate  P elements generally appear to be internal deletions of P factors, and it has been suggested that they arise as a consequence of the mechanics of transposition (Voelker et al. 1984; Daniels, Strausbaugh and Armstrong 1985; Daniels et al. 1985; Engels et al. 1990). If transposition is a function of the number of P factors per genome, then as the factors accumulate (see below), transposition rates might increase dramatically. Consequently, the probability of generating appropriately deleted P elements would also increase. Once individuals having cytotype generating elements appear in the population they might have a selective advantage over their dysgenic siblings. This could explain the observation that once P cytotype begins to appear, the populations acquire it rapidly. An alternate model for the generation of P cytotype suggests that it is the location of the element within the genome, rather than its actual structure, which conveys the regulatory ability (Biemont et al. 1990). It had previously been suggested that genomic position might play a role in the determination of P cytotype (Engels 1989) and for the repression associated with KP elements (Jackson, Black, and Dover 1988). Biemont and colleagues (1990) demonstrate that the presence of a putative complete P element in region 1A of the X chromosome is associated with P cytotype within inbred lines studied. Although this result could be due to identity by descent, M cytotype lines of the same origin had no insertions in 1A. Additionally, region 1A had previously been shown to be a hotspot for P element insertions in natural populations (Ajioka and Eanes  47  1989) and this site was found to be more common in populations of P cytotype than those of M cytotype (Ronsseray, Lehmann and Anxolabehere 1989). The distal portion of an X chromosome that contains two P elements at site 1A has recently been isolated in a strain that is otherwise devoid of P elements (Ronsseray, Lehmann and Anxolabehere 1991). At least one of these elements produces transposase, yet this strain exhibits all of the characteristics of P cytotype including maternal inheritance of the ability to repress transposase activity in the germline. When somatic transposase activity is provided by a modified P element, this strain can also partially regulate this activity. However, this regulatory ability is chromosomally inherited. These results are interpreted to suggest that the regulatory elements are inserted near a germline-specific enhancer. Although these regulatory elements could represent a special class of modified elements, it is also possible that they could be complete elements. Biemont  et al (1990) hypothesize that altered transcription levels of P elements.  could have some secondary effect on the splicing of the third intron resulting in the production of a repressor rather than a transposase. In either case, if a few sites which confer P cytotype expression exist in the genome, then it is likely that insertion at these sites might not occur immediately. These proposals are therefore consistent with the observed delay in acquisition of P cytotype. Any model for P cytotype requires some attribute to account for the observed limited maternal inheritance which is ultimately dependent on the presence of chromosomal elements. Several suggestions have been made. A  48  repressor present in the cytoplasm of the egg might positively regulate its own production (O'Hare and Rubin 1983). Alternatively, repressor encoding elements with some capacity for self-replication might exist extrachromosomally (Engels 1981a). Attempts to locate such elements have not been successful. Yet another possibility is that the maternal inheritance of P strains is a result of P elements being inserted adjacent to genes normally expressed during oogenesis (Robertson and Engels 1989; Misra and Rio 1990). Information in these flanking sequences might cause repressor to be synthesized and deposited in the egg during oogenesis. This last model is consistent with the observation that a significant number of genes introduced by P element transformation exhibit expression during oogenesis (Grossniklaus et al. 1989). Any one of these proposals or some combination of them seems plausible given the present knowledge.  Dispersal of P Elements: The single fly ovary blots (Figures 2-2 and 2-3; summarized in Figures 2-4 and 2-5), show that there is a rapid increase in the number of flies containing some P sequences. This dispersal of P elements to new genomes is not surprising. In fact, it would be expected for any element present in high copy number and distributed throughout the genome. One need not invoke replicative transposition or mobilization of any type. Recombination and chromosome assortment would disperse such a high copy number element from a few individuals to many in only a few generations of outbreeding. However, if such simple processes were solely  49  responsible for the spread of P elements, then one would predict that the increase in the frequency of genomes with some elements would be accompanied by a corresponding decrease in the number of elements within individual genomes (see discussion by Anxolabehere et al. 1986; Good and Hickey 1987). Alternatively, if some multiplicative process also occurred, then the number of elements within individual flies might not be reduced.  Accumulation of P Elements and Models for Transposition: There are several lines of evidence which support the latter alternative. First, the dot blot assays on pooled flies (Figures A-3 and A-4) indicate that the total amount of P element hybridizing DNA in the populations increases with increasing generation. Second, the single fly ovary blots suggest that the number of P elements per individual increases in later generations (Figures 2-2 and 2-3). If outbreeding was wholly responsible for P element dispersal, one would expect the intensity of the hybridization per individual fly to decrease with increasing generation. Such a trend is seen in the early generations, however, in later generations this trend is reversed. Finally, the single fly Southern blots also indicate that the number of elements in individual flies increases considerably in the later generations. For 5% population A (Figure 2-6), all flies tested from generation 6 show fewer bands than the 72 control; by generation 14 some of the flies have about as many bands as the control; and by generation 20 the number of bands and the intensity of labelling of all flies tested are at least as great as  50  the control. Similar trends can be seen in Figures 2-7 which contains DNA of flies from generations 6, 14 and 20 of 5% populations B, although the flies from generation 14 appear to have more elements. The decrease in the intensity of the positive ovary blots and decrease in number of bands in the Southern blots of P element containing flies from early generations relative to the initiating 72 flies indicate that chromosome assortment and recombination during outbreeding are important to the dispersal of P elements. This is a significant result because spread experiments done by other investigators without the utilization of molecular assays or with the utilization of transformed lines were unable to demonstrate this initial reduction in the number of P sequences per individual fly. In the early generations after invasion, assortment and recombination probably act quickly to increase the number of individuals with P elements, while reducing the average number of P elements per genome. This does not imply that a multiplicative process does not act in the first generations after invasion. In fact the data in Figures A-3 and A-4 show that there is a continuous increase in the total amount of P element DNA in the populations from generation 2 onwards. It seems likely that outbreeding contributes most to P element dispersal in early generations. In later generations, all the molecular data show that there is an increase in the number of P elements per genome, suggesting that a multiplicative process is counteracting any tendency towards decreasing the number of P elements per genome produced by outbreeding.  51  There are at least four alternatives which might explain the increase in the number of elements accompanying their spread through the populations. The first possibility is that the chromosome segments containing P sequences may be disproportionately represented in the offspring of P-M crosses due to some form of positive selection. This selection could act on the P elements themselves or on associated loci. There is, however, no evidence that P elements confer any selective advantage on flies which contain them. To the contrary, there is evidence that the presence of these elements generates a significant genetic load (Mackay 1986; Fitzpatrick and Sved 1986). Although it would be difficult to exclude the possibility of weak selection for P elements or linked loci, it seems unlikely that selection could account for the dramatic increase in P element number observed in our experiments. Other research has indicated that the rate of transposition can be as much as 1000X the rate of excision (see Engels et al. 1990). A second possibility is that the multiplication of P sequences is due to replicative transposition in which elements undergo DNA synthesis resulting in duplicate elements which then insert elsewhere in the genome. This model is consistent with the single fly Southern analysis. As mentioned above, by generation 20 the number of P homologous bands and the intensity of labelling among individual flies from the experimental populations are at least as great as those of the 72 strain which was used to invade the true M populations. In addition, note that some but not all of the P element homologous bands are shared  52  either between individuals of the experimental group or with the 72 control. This suggests that there are more elements in later generations than the earlier generations in the experimental group, and that many of these elements are located at new positions within the individual genomes. That is, the elements are both multiplying and transposing. The final two plausible mechanisms that could result in the multiplication of P sequences involve conservative cut-and-paste transposition. In this type of transposition the element is cut out of the donor site and pasted into the target DNA. This simple conservative transposition coupled with chromosomal assortment can lead to genotypes with increased numbers of transposable elements. Like unequal crossing-over within gene families, however, this process is expected to be unbiased. It would generate genotypes with an increased or a reduced number of elements with equal likelihood. The observed increase in the mean amount of P hybridizing DNA in all populations indicates a strong positive bias and thus argues against this explanation of the increase in element number. The alternative way that cut-and-paste transposition could result in a multiplication of P elements is if the transposition was followed by double-strand gap repair of the donor chromosome. This repair would involve the broken ends invading an homologous duplex, followed by DNA synthesis, and resolution. Engels et al. 1990 have proposed such a mechanism for P element transposition. The consequence of the double-strand gap repair depends on the information copied into the gap and is thus dependent on the template used for the repair  53  process. If the homologous chromosome is used as a template and it does not contain an element, then repair would yield a precise excision. On the other hand, if the sister chromatid is used as a template or if the homologous chromosome also contains an element, then repair would generate a chromosome identical to the original one. Such a combination of cut-and-paste transposition and double-strand gap repair could thus yield a result identical to that obtained via replicative transposition; a donor site that retains a P element and a target site that obtains one. Obviously the single fly Southern analysis is equally consistent with cut-and-paste transposition followed by double-strand gap repair as it is with replicative transposition. There are two methods of replicative transposition that are known to occur, however, both of these have trouble explaining certain aspects of P element biology (Engels 1989; Engels et al 1990). One method utilizes reverse transcription and a RNA intermediate, while the other utilizes a cointegrate structure (see Berg and Howe 1989). Neither of these models can explain the observation of internally deleted elements or the high frequency of precise excisions that are exceedingly rare for known replicative elements. Reverse transcription via an RNA intermediate would also have difficulty reproducing the entire P elements due to the presence of introns and the absence of long terminal repeats. No full length transcripts have been detected (Laski, Rio and Rubin 1986). Finally, a cointegrate model would predict a high frequency of successive two point chromosome rearrangements with new P elements always generated at  54  the breakpoints. Such new elements are not consistently generated and the rearrangements observed are more consistent with a random breakage and rejoining of chromosomes (Engels and Preston 1984). The model of transposition proposed by Engels et al. (1990),involving cutand-paste transposition followed by gap repair, better explains the observations above. It would predict the types of rearrangements that are seen. The internally deleted elements could simply be the result of a failure to complete the repair process. The two partially extended 3' ends might pair with each other at points of weak homology such as would be provided by short direct duplications within the element. A preference for such repeats as sites of deletion breakpoints has been observed (O'Hare and Rubin 1983; Rio, Laski and Rubin 1986). Engels et al. were drawn to their model by the discovery that a high frequency of precise excision could accompany P element transposition only when a wild-type (no insert) homolog was present. They provided support for the model by demonstrating that, like gene conversion, the frequency of the excisions are highly influenced by the ability of the homologs to pair in the region of the donor site. Inhibition of pairing using a multiply inverted balancer chromosome reduced the excision about four-fold. When the only homologous DNA was an ectopic gene inserted by P element transformation, the excision rate was further reduced but still significantly greater than the rate when no wild-type copy was available. This proposed mechanism of transposition is currently the most acceptable since it is compatible with most of the data.  55  If transposition is important to the spread of P elements then the observed lag in cytotype switching is probably also important. Transposition occurs minimally in populations in which all of the individuals have a P cytotype. In order for the elements to disperse and accumulate they must first undergo a phase of transpositional activity before producing the P cytotype. The P cytotype could then act as a form of self-regulation by preventing over-replication. This would limit the genetic load on the population caused by the P elements (Hickey 1982; Charlesworth and Langley 1986). A balanced system such as this would be assured if P factors were necessary for the accumulation of P cytotype conferring elements, as they appear to be for the KP element accumulation (Black et al. 1987).  Conclusion: Our results suggest that chromosome assortment and recombination play an important role in the dynamics of P element invasion. Some form of transposition that results in multiplication of P element sequences must also be involved. Through a combination of these effects, mixed populations initiated with low frequencies of P genomes became predominantly P type within a few generations. This suggests that even a few migrants could spread P elements very efficiently among natural populations of Drosophila melanogaster. Semiisolated populations, which might remain genetically distinct for non-transposable genetic markers, could thus be rapidly invaded by these mobile DNA sequences.  56  REFERENCES Ajioka, J.W. and W.F. Eanes. 1989. 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Kolesnikov. 1990. A new transposable element in Chironomus thummi. Mol. Gen. Genet. 222: 311-316. Yamamoto, A., F. Hihara, and T.K. Watanabe. 1984. Hybrid dysgenesis in Drosophila melanogaster: Pre-dominance of Q factor in Japanese populations and its change in the laboratory. Genetica 63: 71-77. Young, M.W. 1979. Middle repetitive DNA: A fluid component of the Drosophila genome. Proc. Natl. Acad. of Sci. (USA) 76: 6274-6278.  65    BIOGRAPHICAL INFORMATION  G  NAME:  111-6 STr- 19\  MAILING ADDRESS: (4)  G  LP/AKE L sT  V I:\ NC__ 0 v E riec  PLACE AND DATE OF BIRTH: n/  EDUCATION (Colleges and Universities attended, dates, and degrees):  o k(AnL,, coon Col elq... TV/?ci o 1-/ li c) i'llouli A Aelc i (	 C-- Ile , ( A 6 C 2. - le co )S S c. e) '  53 tit:-  -  -  ,  ,-  ,  (A  GI :. I :r,f ,  , ,)  <e c. —  P C...  POSITIONS HELD: (.)., i:c; Ic).' L  3i  T 1.\  1C-di  -  P. eS..e (,ci^  t\s-S S ‘ (•-4, \ ,  ..  -.-  ,  c...,c.,10e --: . -  :.) 7  PUBLICATIONS (if necessary, use a second sheet): occS; 	c,  is4c.r  . 14, C) L.) -"k ,  cAtn)  ,-  fl\:_cj  +)  (2, }- e.,,rne„.+1  S  12_  AWARDS:  Complete one biographical form for each copy of a thesis presented to the Special Collections Division, University Library. DES  •"7,  0.11-  


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